The First Identification of Carbohydrate Binding Modules Specific to Chitosan

Two carbohydrate binding modules (DD1 and DD2) belonging to CBM32 are located at the C terminus of a chitosanase from Paenibacillus sp. IK-5. We produced three proteins, DD1, DD2, and tandem DD1/DD2 (DD1+DD2), and characterized their binding ability. Transition temperature of thermal unfolding (T_m) of each protein was elevated by the addition of cello-, laminari-, chitin-, or chitosan-hexamer (GlcN)₆. The T_m elevation (ΔT_m) in DD1 was the highest (10.3 °C) upon the addition of (GlcN)₆ and was markedly higher than that in DD2 (1.0 °C). A synergistic effect was observed (ΔT_m = 13.6 °C), when (GlcN)₆ was added to DD1+DD2. From isothermal titration calorimetry experiments, affinities to DD1 were not clearly dependent upon chain length of (GlcN)_n; ΔG_r° values were −7.8 (n = 6), −7.6 (n = 5), −7.6 (n = 4), −7.6 (n = 3), and −7.1 (n = 2) kcal/mol, and the value was not obtained for GlcN due to the lowest affinity. DD2 bound (GlcN)_n with the lower affinities (ΔG_r° = −5.0 (n = 3) ∼ −5.2 (n = 6) kcal/mol). Isothermal titration calorimetry profiles obtained for DD1+DD2 exhibited a better fit when the two-site model was used for analysis and provided greater affinities to (GlcN)₆ for individual DD1 and DD2 sites (ΔG_r° = −8.6 and −6.4 kcal/mol, respectively). From NMR titration experiments, (GlcN)_n (n = 2∼6) were found to bind to loops extruded from the core β-sandwich of individual DD1 and DD2, and the interaction sites were similar to each other. Taken together, DD1+DD2 is specific to chitosan, and individual modules synergistically interact with at least two GlcN units, facilitating chitosan hydrolysis.     

Variable alteration of regional tissue oxygen pressure in rat hippocampus by acute swimming exercise

One of the events in the brain is an increasing cerebral blood flow during exercise. The tissue oxygen level may be increased because blood flow correlates with tissue oxygen level. However, it is little known whether the tissue oxygen pressure in hippocampal region (Hip-pO₂) will be affected by exercise.     

The Ecological Perspective of Microbial Communities in Two Pairs of Competitive Hawaiian Native and Invasive Macroalgae

Marine macroalgae are known to harbor large populations of microbial symbionts, and yet, microbe symbiosis in invasive macroalgae remains largely unknown. In this study, we applied molecular methods to study microbial communities associated with two invasive algae Acanthophora spicifera and Gracilaria salicornia and the two native algae Gracilaria coronopifolia and Laurencia nidifica at spatial and temporal scales in Hawaiian coral reef ecosystems. Bacterial communities of both the invasive and native macroalgae displayed little spatial and temporal variations, suggesting consistent and stable bacterial associations with these macroalgae. Results of this study identified three types of bacterial populations: nonspecific (present in both algal and water samples); algae-specific (found in all algal species); and species-specific (only found in individual species). The bacterial diversity of invasive algae was lower than that of their native counterparts at phylum and species levels. Notably, the vast majority (71 %) of bacterial communities associated with the invasive algae G. salicornia were representatives of Cyanobacteria, suggesting a potential ecological significance of symbiotic Cyanobacteria.     

Sulfation of keratan sulfate proteoglycan reduces radiation-induced apoptosis in human Burkitt’s lymphoma cell lines

This study focuses on clarifying the contribution of sulfation to radiation-induced apoptosis in human Burkitt’s lymphoma cell lines, using 3′-phosphoadenosine 5′-phosphosulfate transporters (PAPSTs). Overexpression of PAPST1 or PAPST2 reduced radiation-induced apoptosis in Namalwa cells, whereas the repression of PAPST1 expression enhanced apoptosis. Inhibition of PAPST slightly decreased keratan sulfate (KS) expression, so that depletion of KS significantly increased radiation-induced apoptosis. In addition, the repression of all three N-acetylglucosamine-6-O-sulfotransferases (CHST2, CHST6, and CHST7) increased apoptosis. In contrast, PAPST1 expression promoted the phosphorylation of p38 MAPK and Akt in irradiated Namalwa cells. These findings suggest that 6-O-sulfation of GlcNAc residues in KS reduces radiation-induced apoptosis of human Burkitt’s lymphoma cells.     

Discovery of a disused desaturase gene from the pheromone gland of the moth Ascotis selenaria, which secretes an epoxyalkenyl sex pheromone

Female Ascotis selenaria (Geometridae) moths use 3,4-epoxy-(Z,Z)-6,9-nonadecadiene, which is synthesized from linolenic acid, as the main component of their sex pheromone. While the use of dietary linolenic or linoleic fatty acid derivatives as sex pheromone components has been observed in moth species belonging to a few families including Geometridae, the majority of moths use derivatives of a common saturated fatty acid, palmitic acid, as their sex pheromone components. We attempted to gain insight into the differentiation of pheromone biosynthetic pathways in geometrids by analyzing the desaturase genes expressed in the pheromone gland of A. selenaria. We demonstrated that a Δ11-desaturase-like gene (Asdesat1) was specifically expressed in the pheromone gland of A. selenaria in spite of the absence of a desaturation step in the pheromone biosynthetic pathway in this species. Further analysis revealed that the presumed transmembrane domains were degenerated in Asdesat1. Phylogenetic analysis demonstrated that Asdesat1 anciently diverged from the lineage of Δ11-desaturases, which are currently widely used in the biosynthesis of sex pheromones by moths. These results suggest that an ancestral Δ11-desaturase became dysfunctional in A. selenaria after a shift in pheromone biosynthetic pathways.     

The transition of mouse pluripotent stem cells from the naïve to the primed state requires Fas signaling through 3-O sulfated heparan sulfate structures recognized by the HS4C3 antibody

The characteristics of pluripotent embryonic stem cells of human and mouse are different. The properties of human embryonic stem cells (hESCs) are similar to those of mouse epiblast stem cells (mEpiSCs), which are in a later developmental pluripotency state, the so-called “primed state” compared to mouse embryonic stem cells (mESCs) which are in a naïve state. As a result of the properties of the primed state, hESCs proliferate slowly, cannot survive as single cells, and can only be transfected with genes at low efficiency. Generating hESCs in the naïve state is necessary to overcome these problems and allow their application in regenerative medicine. Therefore, clarifying the mechanism of the transition between the naïve and primed states in pluripotent stem cells is important for the establishment of stable methods of generating naïve state hESCs. However, the signaling pathways which contribute to the transition between the naïve and primed states are still unclear. In this study, we carried out induction from mESCs to mEpiSC-like cells (mEpiSCLCs), and observed an increase in the activation of Fas signaling during the induction. The expression of Fgf5, an epiblast marker, was diminished by inhibition of Fas signaling using the caspase-8 and -3 blocking peptides, IETD and DEVD, respectively. Furthermore, during the induction, we observed increased expression of 3-O sulfated heparan sulfate (HS) structures synthesized by HS 3-O-sulfotransferase (3OST), which are recognized by the HS4C3 antibody (HS4C3-binding epitope). Knockdown of 3OST-5 reduced Fas signaling and the potential for the transition to mEpiSCLCs. This indicates that the HS4C3-binding epitope is necessary for the transition to the primed state. We propose that Fas signaling through the HS4C3-binding epitope contributes to the transition from the naïve state to the primed state.     

An Ascochlorin Derivative,AS-6, Potentiates Insulin Action in Streptozotocin Diabetic Mice and Rats

γ-Aminobutyric acid (GABA), a hypotensive compound, and alanine accumulated in tea leaves under anaerobic conditions. Since the ¹⁵N in ¹⁵N-glutamic acid was well incorporated in GABA and alanine during anaerobic incubation, glutamic acid seemed to be a source of nitrogen for the increased GABA and alanine. GOT and GPT were the predominant amino acid transaminases in tea leaves. Although glutamate decarboxylase and GPT seemed to be important for GABA and alanine accumulation, the activities of these enzymes did not increase under anaerobic conditions. Glutamate decarboxylase, which formed GABA from glutamate, was purified 52.4-fold. This enzyme, with an optimum pH at 5.8, was activated by pyridoxal phosphate and used only l-glutamic acid as a substrate.     

Comparison of Mineral Balances in Germfree and Conventional Mice When Sodium Phytate is Added to Purified Diet

A strain of Alcaligenes isolated from soil was a good producer of β-glucuronidase, and the enzyme was purified from the cell-free extract by sequential column chromatography on DEAE-Toyopearl, Toyopearl HW-55F, and Phenyl-Sepharose CL-4B. By these procedures, two β-glucuronidases designated as β-glucuronidases I and II were purified 240- and 508-fold, respectively. β-Glucuronidase I, with a molecular weight of 75,000, had an optimum pH at 7.5 and the enzyme II, with a molecular weight of 300,000, had maximum activity at pH 6.0. Both enzymes were strongly inhibited by saccharo-1,4-lactone, glucaro-δ-lactam, p-chloromercuribenzoate, Hg²⁺, and N-bromosuccinimide. β-Glucuronidase I was active toward estrogen-3-β-glucuronides and inert toward β-glucuronide conjugates of menthol, estrogen-17β-, estrogen-16α-, androsterone-3α-, testosterone-17β-, cortisol-17α-. β-Glucuronidase II hydrolyzed all of these substrates. β-Glucuronidase I was inhibited by phenolphthalein and its glucuronide.     

5,7-Dodecadien-1-ol: Candidate for the Sex Pheromone of the Pine Moth

Ferredoxin-nitrite reductase (EC 1.7.7.1), an enzyme which catalyzes the 6-electron reduction of nitrite to ammonia, has been isolated from Spinacia oleracea. The isolated enzyme was homogeneous by disc electrophoresis with polyacrylamide gel. The molecular weight of the enzyme was estimated to be 86,000 by Ultrogel AcA 34 gel filtration. In the oxidized form, the enzyme had absorption maxima at 278, 388 (Soret band), 573 (α band) and 690 nm, indicating that siroheme is directly involved in the catalysis of nitrite reduction. This absorption spectrum was modified by sulfite, hydroxylamine and cyanide. The enzyme exhibited electron paramagnetic resonance signals with g values of 6.9 and 5.2, which are characteristic of a high spin Fe³⁺ -siroheme in the molecule. These signals disappeared upon the addition of dithionite or nitrite. This isolated enzyme also contained four moles of labile sulfide and 7 g-atoms of iron per 86,000 g of protein.